Bone marrow transplantation following supralethal radiochemotherapy is associated with dangerous infections due to the slow immune reconstitution during the first year post transplant. Thus, the use of reduced intensity conditioning, associated with less severe immune ablation, could have a remarkable potential in the treatment of a variety of non-malignant diseases, or for the induction of 'mixed chimerism' as a prelude for cell therapy in cancer or in organ transplantation. However, the marked level of host hematopoioetic and immune cells surviving mild preparatory regimens represents a difficult barrier for the engraftment of donor cells. In particular, the use of purified allogeneic stem cells, which do not pose any risk for GvHD and which can continuously present donor type antigens in the host thymus, thereby inducing durable tolerance to donor cells or tissues, represents one of the most desirable goals in transplantation biology. The proposed project will address this challenge by investigating the potential of early myeloid veto cells generated upon short term culture of hematopoietic stem cells, to allow engraftment of mega dose purified stem cells following reduced intensity conditioning. Initially, the optimal veto cell numbers which can overcome T cells mediated graft rejection will be defined in a mouse model specifically developed for this purpose (aim 1a). Subsequently, the potential synergism of veto CTLs with rapamycin (aim 1b) will be tested in the same model. Upon completion of this study the minimal rapamycin dose and the minimal number of veto CTLs and which lead, together, to durable engraftment of allogeneic stem cells will be defined in recipient mice following reduced intensity conditioning (aim 1c). The protocols of choice developed in 1c will be evaluated for their contribution to the immune competence of the resulting chimera, compared to full donor type chimera achieved by stem cell transplantation in fully ablated hosts (aim 2a). In particular, the immune response of chimeric mice to a challenge of murine CMV infection, at different times post transplant, will serve as an important endpoint prior to clinical application (aim 2b). In parallel to the engraftment studies, the mechanism of action by which early hematopoietic stem cells can paralyze immune responses against their alloantigens, will be investigated. By using different knockout mice, the role of TNF and TNF related molecules will defined (aim 3a). Gene chip murine high-density oligonucleotide arrays, representing 6,500 murine genes will be used to discover potential apoptosis pathways induced upon the recognition of mouse hematopoietic stem cells by alloreactive T cells (aim 3b). Based on insights on the mechanism of action of veto stem cells from aim 3a and aim 3b, new synergistic agents will be further assessed and developed in the mouse model (aim 3c).